KCTD Hetero-oligomers Confer Unique Kinetic Properties on Hippocampal GABAB Receptor-Induced K+ Currents.

GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABAB receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K+-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABAB receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K+ currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABAB receptors and modulates physiologically induced K+ current responses in the hippocampus. SIGNIFICANCE STATEMENT: The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.

Patient-reported symptoms during radiotherapy : Clinically relevant symptom burden in patients treated with palliative and curative intent.

BACKGROUND: The benefits of patient-reported symptom assessment combined with integrated palliative care are well documented. This study assessed the symptom burden of palliative and curative-intent radiation oncology patients. PATIENTS AND METHODS: Prior to first consultation and at the end of RT, all adult cancer patients planned to receive fractionated percutaneous radiotherapy (RT) were asked to answer the Edmonton Symptom Assessment Scale (ESAS; nine symptoms from 0 = no symptoms to 10 = worst possible symptoms). Mean values were used for curative vs. palliative and pre-post comparisons, and the clinical relevance was evaluated (symptom values ≥ 4). RESULTS: Of 163 participating patients, 151 patients (90.9%) completed both surveys (116 curative and 35 palliative patients). Before beginning RT, 88.6% of palliative and 72.3% of curative patients showed at least one clinically relevant symptom. Curative patients most frequently named decreased general wellbeing (38.6%), followed by tiredness (35.0%), anxiety (32.4%), depression (30.0%), pain (26.3%), lack of appetite (23.5%), dyspnea (17.8%), drowsiness (8.0%) and nausea (6.1%). Palliative patients most frequently named decreased general wellbeing (62.8%), followed by pain (62.8%), tiredness (60.0%), lack of appetite (40.0%), anxiety (38.0%), depression (33.3%), dyspnea (28.5%), drowsiness (25.7%) and nausea (14.2%). At the end of RT, the proportion of curative and palliative patients with a clinically relevant symptom had increased significantly to 79.8 and 91.4%, respectively; whereas the proportion of patients reporting clinically relevant pain had decreased significantly (42.8 vs. 62.8%, respectively). Palliative patients had significantly increased tiredness. Curative patients reported significant increases in pain, tiredness, nausea, drowsiness, lack of appetite and restrictions in general wellbeing. CONCLUSION: Assessment of patient-reported symptoms was successfully realized in radiation oncology routine. Overall, both groups showed a high symptom burden. The results prove the need of systematic symptom assessment and programs for early integrated supportive and palliative care in radiation oncology.

Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.

Identification of Disparities in Personalized Cancer Care-A Joint Approach of the German WERA Consortium.

(1) Background: molecular tumor boards (MTBs) are crucial instruments for discussing and allocating targeted therapies to suitable cancer patients based on genetic findings. Currently, limited evidence is available regarding the regional impact and the outreach component of MTBs; (2) Methods: we analyzed MTB patient data from four neighboring Bavarian tertiary care oncology centers in Würzburg, Erlangen, Regensburg, and Augsburg, together constituting the WERA Alliance. Absolute patient numbers and regional distribution across the WERA-wide catchment area were weighted with local population densities; (3) Results: the highest MTB patient numbers were found close to the four cancer centers. However, peaks in absolute patient numbers were also detected in more distant and rural areas. Moreover, weighting absolute numbers with local population density allowed for identifying so-called white spots-regions within our catchment that were relatively underrepresented in WERA MTBs; (4) Conclusions: investigating patient data from four neighboring cancer centers, we comprehensively assessed the regional impact of our MTBs. The results confirmed the success of existing collaborative structures with our regional partners. Additionally, our results help identifying potential white spots in providing precision oncology and help establishing a joint WERA-wide outreach strategy.

Patient-Reported Outcomes-Secondary Analysis of the SCORE-2 Trial Comparing 4 Gy × 5 to 3 Gy × 10 for Metastatic Epidural Spinal Cord Compression.

PURPOSE: To compare 4 Gy × 5 (1 week) to 3 Gy × 10 (2 weeks) in relieving pain and distress in patients with metastatic epidural spinal cord compression (MESCC). METHODS AND MATERIALS: The randomized SCORE-2 trial compared 4 Gy × 5 (n = 101) to 3 Gy × 10 (n = 102) for MESCC. In this additional analysis, these regimens were compared for their effect in relieving pain and distress. Distress was evaluated with the distress-thermometer (0 = no distress, 10 = extreme distress) and pain on a linear scale (0 = no pain, 10 = worst pain). Relief of distress was defined as decrease of ≥2 points; complete and partial pain relief were defined as achieving a score of 0 points and a decrease ≥2 points, respectively, without increase of analgesic use. This prospective secondary analysis of the SCORE-2 trial aimed to show that 4 Gy × 5 was not inferior to 3 Gy × 10 regarding distress and pain relief. Analyses were performed using the unconditional test of noninferiority for binomial differences based on restricted maximum likelihood estimates (noninferiority margin: -20%). Evaluations were performed before, directly after, and 1, 3, and 6 months after radiation therapy. (ClinicalTrials.gov: NCT02189473). RESULTS: At baseline, median distress scores were 8 (2-10) points in the 4 Gy × 5 group and 8 (2-10) points in the 3 Gy × 10 group. At 1 month, distress relief rates were 58.1% (43/74) and 62.7% (47/75) (difference: -4.6%; 95% confidence interval, -20.0% to +11.1%; P = .025). At baseline, median pain scores were 7 (2-10) and 7 (2-10) points, respectively. At 1 month, complete pain relief rates were 23.5% (16/68) versus 20.0% (14/70) (difference, +3.5%; 95% confidence interval, -10.4% to +17.5%; P < .001), and overall pain relief rates were 52.9% (36/68) versus 57.1% (40/70) (difference, -4.2%; 95% confidence interval, -20.5% to +12.3%; P = .029). Distress and pain relief rates after 4 Gy × 5 were largely comparable to 3 Gy × 10 at all time points. Associated 95% confidence intervals did not point toward any relevant differences. CONCLUSIONS: In patients with MESCC and poor to intermediate survival prognoses, 4 Gy × 5 appeared noninferior to 3 Gy × 10 regarding pain and distress relief.

Comparison of Two Radiotherapy Regimens for Metastatic Spinal Cord Compression: Subgroup Analyses from a Randomized Trial.

BACKGROUND/AIM: According to our randomized trial, 5×4Gy was comparable to 10×3Gy for metastatic spinal cord compression. Since it remained unclear whether findings applied to poor and intermediate prognoses patients, subgroup analyses were performed. PATIENTS AND METHODS: In patients with poor prognoses, 58 received 5×4Gy, 53 received 10×3Gy. In intermediate-prognoses patients, numbers were 43 and 49. RESULTS: In patients with poor prognoses, 1-month overall response (OR) was 85% after 5×4Gy and 10×3Gy (p=0.99), improvement 38% vs. 42%, ambulatory status 60% vs. 64% (p=0.83), 6-month local progression-free survival (LPFS) 75% vs. 69% (p=0.74) and 6-month overall survival (OS) 26% vs. 19% (p=0.43). In patients with intermediate prognoses, 1-month OR was 89% after 5×4Gy and 93% after 10×3Gy (p=0.85), improvement 39% vs. 45%, ambulatory status 84% vs. 82% (p=0.90), 6-month LPFS 79% vs. 92% (p=0.17) and 6-months OS 65% vs. 58% (p=0.65). CONCLUSION: 5×4Gy was not significantly inferior to 10x3Gy in both subgroups.

Results of a multicenter study investigating the potential impact of the overall treatment time on outcomes of radiation therapy alone with 5×4 Gy for metastatic epidural spinal cord compression.

PURPOSE/OBJECTIVE: A 5×4 Gy program is commonly used for metastatic epidural spinal cord compression (MESCC). It is unclear whether an overall treatment time (OTT) of 5 days (5 consecutive fractions) results in better outcomes than an OTT of 7 days (ie, no irradiation during the weekend). METHODS AND MATERIALS: A total of 111 patients who received 5×4 Gy over 5 consecutive days were retrospectively compared with 277 patients treated with 5×4 Gy over 7 days (no irradiation during the weekend) for effect on motor function, local control of MESCC, and overall survival (OS). Ten further characteristics were evaluated: age, gender, interval tumor diagnosis to MESCC, visceral metastases, other bone metastases, primary tumor type, time developing motor deficits, walking ability, vertebrae involved, and performance status. RESULTS: On multivariate analysis regarding post-radiation therapy motor function, primary tumor type (P = .011) and time developing motor weakness (P < .001) were significant, whereas the OTT did not even achieve significance on univariate analysis (P = .99). On multivariate analysis of local control, visceral metastases (P = .006) were significant. Again, the OTT was not even significant on univariate analysis (P = .81). On multivariate analysis of OS, interval tumor diagnosis to MESCC (P = .015), visceral metastases (P .001), tumor type (P = .003), walking ability (P < .001), and Eastern Cooperative Oncology Group performance score (P < .001) achieved significance. Even on univariate analysis, OTT did not have an effect on OS (P = .79). CONCLUSIONS: Longer OTT did not impair outcomes of irradiation with 5×4 Gy for MESCC; thus, no compensation (for example an additional radiation fraction) is necessary if the radiation treatment is not continued during the weekend.

Role of the overall treatment time of radiotherapy with 10 × 3 Gy for outcomes in patients with metastatic spinal cord compression.

INTRODUCTION: Radiotherapy alone still is the most commonly delivered treatment modality for metastatic spinal cord compression (MSCC). MSCC is an emergency situation, which requires treatment as soon as possible. When radiotherapy is performed with the most commonly used approach 10 × 3 Gy (30 Gy in 10 fractions) over 2 weeks, the question has been asked whether an overall treatment time (OTT) of 14-15 days including two weekends without irradiation leads to worse outcomes than OTT of 12 days (beginning of radiotherapy on a Monday resulting in only one weekend break)? METHODS: A total of 412 patients with MSCC were included in this retrospective study. Ninety-two patients receiving 10 × 3 Gy over 12 days were compared to 320 patients with an OTT of 14-15 days. Ten additional factors were investigated. RESULTS: On multivariate analysis, functional outcome was associated with tumour type (P < 0.001), time developing motor deficits (P < 0.001), ambulatory status (P = 0.018) and performance score (P < 0.001); OTT had no significant impact (P = 0.40). On univariate analysis of local control of MSCC (freedom from recurrence in irradiated spinal parts), no factor was significant including OTT (P = 0.66). On multivariate analysis, visceral metastases (P < 0.001), tumour type (P < 0.001), time developing motor deficits (P < 0.001), ambulatory status (P < 0.001) and performance score (P < 0.001) were associated with survival, OTT not even on univariate analysis (P = 0.55). CONCLUSIONS: Since an OTT of 14-15 days had no negative impact on outcomes compared to 12 days, compensation in form of an additional radiation fraction or continuation of radiotherapy during weekends is not required, if radiotherapy cannot be started on a Monday.

Radiotherapy With 4 Gy × 5 Versus 3 Gy × 10 for Metastatic Epidural Spinal Cord Compression: Final Results of the SCORE-2 Trial (ARO 2009/01).

PURPOSE: To compare short-course radiotherapy (RT) (4 Gy × 5) to longer-course RT (3 Gy × 10) for metastatic epidural spinal cord compression (MESCC). PATIENTS AND METHODS: Two-hundred three patients with MESCC and poor to intermediate expected survival were randomly assigned to 4 Gy × 5 in 1 week (n = 101) or 3 Gy × 10 in 2 weeks (n = 102). Patients were stratified according to ambulatory status, time developing motor deficits, and primary tumor type. Seventy-eight and 77 patients, respectively, were evaluable for the primary end point, 1-month overall response regarding motor function defined as improvement or no further progression of motor deficits. Other study end points included ambulatory status, local progression-free survival, and overall survival. End points were evaluated immediately after RT and at 1, 3, and 6 months thereafter. RESULTS: At 1 month, overall response rates regarding motor function were 87.2% after 4 Gy × 5 and 89.6% after 3 Gy × 10 (P = .73). Improvement rates were 38.5% and 44.2%, respectively, no further progression rates 48.7% and 45.5%, respectively, and deterioration rates 12.8% and 10.4%, respectively (P = .44). Ambulatory rates at 1 month were 71.8% and 74.0%, respectively (P = .86). At other times after RT, the results were also not significantly different. Six-month local progression-free survival was 75.2% after 4 Gy × 5 and 81.8% after 3 Gy × 10 (P = .51); 6-month overall survival was 42.3% and 37.8% (P = .68). CONCLUSION: Short-course RT with 4 Gy × 5 was not significantly inferior to 3 Gy × 10 in patients with MESCC and poor to intermediate expected survival.

Distribution of the auxiliary GABAB receptor subunits KCTD8, 12, 12b, and 16 in the mouse brain.

GABA(B) receptors are the G-protein-coupled receptors for γ-aminobutyric acid (GABA). KCTD8, 12, 12b, and 16 were recently identified as auxiliary GABA(B) receptor subunits and distinctly influence biophysical and pharmacological properties of the receptor response. Here we examined the expression patterns of the KCTDs in the mouse brain. Using in situ hybridization analysis, we found that most neurons express KCTD transcripts, supporting biochemical data showing that most GABA(B) receptors in the brain incorporate KCTD proteins. In the adult brain, KCTD12 and 16 have a widespread and KCTD8 and 12b a restricted expression pattern. Individual neurons can coexpress multiple KCTDs, as shown for granule cells and CA1/CA3 pyramidal cells in the hippocampus that coexpress KCTD12 and 16. In contrast, granule, Purkinje, and Golgi cells in the cerebellum selectively express one KCTD at a time. The expression levels of individual KCTD transcripts vary during postnatal brain development. Immunohistochemistry reveals that individual KCTD proteins can exhibit distinct axonal or dendritic localizations in neuronal populations. KCTDs are also detectable in nonneuronal tissues not expected to express GABA(B) receptors, suggesting that the role of KCTD proteins extends beyond GABA(B) receptors. In summary, our findings support that most brain GABA(B) receptors associate with KCTD proteins, but that the repertoire and abundance of KCTDs varies during development, among brain areas, neuronal populations, and at subcellular sites. We propose that the distinct spatial and temporal KCTD distribution patterns underlie functional differences in native GABA(B) responses.

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function.

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.

Klebsiella oxytoca as an agent of antibiotic-associated hemorrhagic colitis.

BACKGROUND & AIMS: Klebsiella oxytoca has been isolated from stools and colonic biopsy specimens of patients with Clostridium difficile-negative antibiotic-associated hemorrhagic colitis (AAHC), but the pathogenic role of the germ has not been established. The purpose of this study was to investigate the presence of K. oxytoca in patients with AAHC from a prospective cohort of patients with acute colitis, and to test the cytotoxicity on HEp-2 cells of K. oxytoca strains from patients with AAHC and healthy carriers. METHODS: Colonic biopsy specimens and a sample of colonic fluid from 93 consecutive patients with acute colitis were cultured on selective media for 7 established pathogens and K. oxytoca. The 2 K. oxytoca strains isolated in the 4 patients with C. difficile-negative AAHC of this cohort and 105 additional K. oxytoca strains from patients with C. difficile-negative AAHC (n = 15) and healthy carriers (n = 90) were tested for cytotoxicity using a HEp-2 cell culture assay. RESULTS: K. oxytoca was isolated in 50% (2 of 4) of the patients of the prospective cohort with C. difficile-negative AAHC compared with 2% (1 of 41) of the patients with acute colitis caused by established pathogens (P = 0.02). The rate of cytotoxic strains of K. oxytoca was higher in patients with AAHC (82%) than in healthy carriers (42%, P = 0.003). CONCLUSIONS: We conclude that K. oxytoca is isolated with a significant high rate in patients with C. difficile-negative AAHC, and that K. oxytoca strains from patients with AAHC are cytotoxic more frequently on HEp-2 cells than strains from healthy carriers. These results strengthen the hypothesis of a causative role of K. oxytoca in some of the patients with AAHC.